Effect of calcium concentration against inoculum to substrate ratio
Inoculum to substrate ratio (I/S) had a significant effect on biogas production (Fig. 1). When I/S ratio was lower than 1, the AD process was severely inhibited in the high lipid concentration (NS1) digester. The calcium dosed digester showed enhanced biogas production by 10 % as well as accelerated reaction rate at I/S ratio of 1 in both the NS1 and low lipid concentration (NS2) digesters. At I/S ratio of 1, calcium dosing with calcium to LCFAs ratio of 0.5 noticeably increased methane production while further increase of calcium to LCFAs ratio had barely any effect on biogas production. Actually, a single calcium ion could bond with two LCFA molecules, so that the calcium to LCFA ratio of 0.5 would be ideal if calcium and LCFAs were completely mixed. Further increase in calcium concentration could not bond more LCFAs, leading to no effect on free LCFA concentration.
Calcium had no effect on biogas production when the I/S ratio was extremely low, even with high concentration of calcium. A possible explanation was at such a low inoculum concentration, although calcium was added, the slow methanogenesis step controlled the whole process, which led to VFA accumulation other than LCFAs as the main inhibitor. The same explanation could be applied when the I/S ratio was 0.4: the released and free algal cells with use of high concentration of calcium raised the hydrolytic rate, but with an unmatched increase in methanogenic rate, the system generated accumulated VFA, prolonging the lag phase and low gas production due to VFA inhibition. The high VFA concentration and low pH were also observed by Zhao et al. [2] when digesting algal biomass at low inoculum concentration. It was noticed that LCFA concentration was higher in digesters with calcium addition compared with control, which led to a delay in the degradation of LCFAs for all digesters with calcium dosage (Figs. 2, 3).
The modeled relationship of specific methane production (SMP) with inoculum:LCFA ratio and calcium:LCFA ratio is illustrated in Fig. 4. It can be seen that inoculum concentration had greater effect on SMP than calcium concentration. Sufficient inoculum was extremely important for healthy digestion without inhibition. With high inoculum:LCFA ratio of 1.0, SMP could reach the value reported by [2] (0.56 and 0.38 L CH4/g VS for NS1 and NS2, respectively). Palatsi et al. [21] confirm this observation, detailing that increases in inoculum concentration are the most efficient and fast recovery strategy for an LCFA-inhibited digestion process.
In this research, the LCFA concentration in NS1 and NS2 digesters was 9.9 g COD/L and 3.1 g COD/L, respectively, noticeably higher than the approximate inhibitory threshold range (~0.5–1.5 g COD/L) mentioned in literature [6, 8, 26, 27]. Severe inhibition occurred in digesters with low inoculum concentration, as noticed by extremely low methane production. However, no inhibition was observed for digesters with appropriate I/S ratio and proper calcium dosing. It seems that high inoculum concentration could be used as a mean of alleviating the inhibition mediated by LCFAs. Calcium ion could also be an effective way to bond LCFAs and thus keep microbial cells from being tightly wrapped by LCFAs. The impact of calcium ion, however, is dependent on the concentration of inoculum, in which a minimum inoculum concentration is required.
Kinetic analysis of inhibition on anaerobic digestion of algal biomass
The accumulated methane production curves for NS1 and NS2 at various I/S ratio and calcium concentration were simulated with the developed kinetic model (Figs. 2, 3). The LCFA degradation profiles were then predicted with the developed model.
The inhibition of LCFAs on anaerobic microbial consortia has been kinetically investigated as \(K_{\text{I}}\) with a range of 1.3–3.4 kg COD/m3 [28, 29]. However, the extent of inhibition varies among hydrolytic bacteria, acidogens and methanogens. Thus, a lumped inhibition factor \(K_{\text{I}}\) for whole anaerobic microbial consortium is not sufficient to kinetically describe the different inhibition effect of LCFAs on each microbial group. In this research, the inhibition of LCFAs was evaluated based on individual microbial groups for a more accurate estimation. The results show that inhibition factors for hydrolytic bacteria (\(K_{\text{h}}\)), acidogenic bacteria (\(K_{\text{v}}\)), and methanogens (\(K_{\text{m}}\)) were in the range of 2.6–9.4, 2.1–7.9, and 1.0–2.9 kg COD/m3, respectively (Fig. 4). The data suggested a more severe LCFA inhibition on methanogens than on hydrolytic bacteria and acidogens. As a first time kinetic evidence of LCFA inhibition on different functional groups, methanogenesis could be the rate-limiting step in an LCFA-inhibited digestion process, which is consistent with previous research [9, 11].
The I/S ratio had a remarkable effect on each inhibition factor, with regard to its role in affecting SMP. However, kinetic behavior of each microbial group varies against the change of I/S ratio. As the I/S ratio increased from 0.1 to 1.0, \(K_{\text{h}}\), \(K_{\text{v}}\) and \(K_{\text{m}}\) boosted from 2.6, 2.1 and 1.0 to 8.5, 5.8 and 2.3 kg COD/m3, respectively, when calcium was not added. Apparently, the inhibition factor of hydrolytic bacteria was most affected by inoculum concentration while that of methanogen was less affected.
Calcium ion concentration showed a limited effect on inhibition factors and the effects on each inhibition factor were similar. However, these effects were dependent on I/S ratio. The value of inhibition factors doubled with calcium dosing at low I/S ratio while the impacts of calcium ion concentration were less significant at high I/S ratio.
This is the first research that kinetically investigated individual inhibition factors for hydrolytic bacteria, acidogens and methanogens, respectively, rather than a lumped inhibition factor for whole microbial consortium by LCFAs. In previous research, one inhibition factor was used for all biological process including hydrolysis, acidogenesis and methanogenesis [17, 19, 29, 30]. The LCFA model developed in this study provided new insights regarding dynamics of the LCFA inhibition process and showed a different inhibition level on each function group. Methanogens were the most fastidious group and were severely impacted by LCFAs; thus, methanogenesis could be the rate-limiting step during AD. Although hydrolytic bacteria were inhibited by LCFAs, and were most impacted by I/S ratio, hydrolysis could be considered the fastest step. Acidogens were also inhibited by LCFAs, one of its products, which led to acidogenesis being a self-limiting step. However, under the condition without LCFA inhibition, hydrolysis is still the rate-limiting step in anaerobic digestion of microalgae, in which pretreatment could play a role.
Microbial community structure analysis with Illumina Miseq sequencing
Two samples from digesters fed with NS1 and NS2, respectively, at I/S ratio of 1 without calcium addition as well as original inoculum were subject to microbial community structure analysis. In total, 36,825 bacteria sequences for 3 samples were classified into 591 genera. The difference of phylum distribution was observed between the two digesters. NS1 digester was dominated by Proteobacteria, followed by Chloroflexi, and Firmicutes, while Firmicutes, Bacteroidetes, Chloroflexi, and Proteobacteria were dominant in NS2 digester with balanced abundance. Moreover, Gammaproteobacteria belonging to Phylum Proteobacteria was enriched in both digesters. The genus level identification of the bacteria communities is illustrated in Fig. 5. Bacteria community in original inoculum showed a balanced population with high diversity. Bacteria community in the NS1 digester showed a distinct pattern with domination of Acinetobacter (blue, 39.6 %), Levilinea (red, 7.0 %), Proteiniclasticum (green, 7.7 %), and Stenotrophomonas (purple, 13.9 %). Acinetobacter was reported to be the main strain among several pure cultures degrading lipid-containing wastewater with efficient lipase secretion capability [31, 32]. This correlates well with the domination of Acinetobacter in the NS1 digester. The bacterial community in the NS2 digester was dominated by Levilinea (red, 7.6 %), Tissierella (light blue, 11.9 %), Proteiniclasticum (green, 6.4 %), Clostridium (orange, 7.6 %), and Parabacteroides (dark blue, 11.0 %). The population analysis demonstrates that a clearly different microbial community structure was formed in the two digesters due to different lipids loading, although hydrolytic/acidogenic bacteria dominated both NS1 and NS2 digesters.
Stenotrophomonas is responsible for the hydrolysis and fermentation of carbohydrate and amino acids [33]. Syntrophic acetogens, including Clostridium, Smithella, Tissierella, Syntrophorhabdus, Sedimentibacter and Sporacetigenium, also presented in the two digesters, although the concentrations were low. Interestingly, the abundance of syntrophic acetogens in the NS1 digester (10.8 %) was significantly lower than that in the NS2 digester (28.2 %), suggesting that syntrophic acetogens were more sensitive to high lipid concentration.
Methanogenic archaea communities were analyzed in the three samples, with a total of 14,220 reads affiliated to 15 genera and 3 orders. The genus level identification of the archaea communities is illustrated in Fig. 6. Methanogenic archaea community in the original inoculum was dominated by Methanolinea (purple, 47.0 %), a strict hydrogenotrophic genus, and Methanosaeta (blue, 44.1 %), a strict aceticlastic methanogen genus. However, Methanosaeta (blue) prevailed in both of the communities of the NS1 and NS2 digesters (77.6 and 74.4 %, respectively), followed by two hydrogenotrophic genera, Methanobacterium (red, around 8 %) and Methanomethylovorans (light blue, around 11 %) in both digesters, indicating that aceticlastic methanogenesis was the main pathway for methane formation in the two digesters, regardless of the different lipids content. Dominance of Methanosaeta was also found in the anaerobic reactors treating microalgal biomass which was attributed to the low levels of acetate [34].
Proposed mechanism of calcium mitigated LCFA inhibition
Inhibition of LCFAs could be mainly attributed to physical attachment on the surface of microbial cells. As microbes are coated by LCFAs, limitations on transportation hinder substrate access and subsequent biogas release [17]. Calcium ions could bond free LCFAs, thus reducing the amount of LCFAs available for microbial cells to half of original LCFA concentration (Graphic abstract). This reduction delayed LCFA degradation, compared with the control. Moreover, the steric hindrance effect of calcium bonded LCFAs could further mitigate LCFA inhibition by loosening the LCFA coat. However, calcium ions could not exclusively compete the LCFAs from the surface of microbial cells. As a result, calcium ion addition could not help mitigate LCFA inhibition for those LCFAs already attached on the surface of microbial cells. Moreover, the effect of calcium ion was limited, and it only played a role when the microbial concentration reached a minimal requirement.
It is foreseeable that multivalent ions, ferric ion for example, could bond more LCFAs according to its charge and give rise to a more sophisticated steric hindrance effect, leading to a stronger effect on alleviating LCFA inhibition while using a reduced amount. The optimal multivalent ion to LCFA ratio would be reciprocal to the value of its charge. However, the same rule as calcium still applies, in that it could not relieve LCFAs inhibition after LCFAs attached to the surface of microbial cells.